Method of treating asthma or allergy by administering an IL-33 receptor antibody

ABSTRACT

Provided herein are methods of modulating IL-33 activity, e.g., for the purpose of treating immune diseases and conditions, as well as methods of screening for compounds capable antagonizing IL-33 signaling.

This application is a Division of U.S. patent application Ser. No.11/779,755, filed on Jul. 18, 2007, now U.S. Pat. No. 7,560,530, issuedJul. 14, 2009, which claims benefit of U.S. Provisional PatentApplication Nos. 60/832,256 (filed Jul. 20, 2006), 60/835,250 (filedAug. 3, 2006), and 60/887,257 (filed Jan. 30, 2007), each of which ishereby incorporated by reference in their entireties.

The Sequence Listing filed electronically herewith is also herebyincorporated by reference in its entirety (File Name:BP06497US02_SeqListing.txt; Date Created: Jul. 10, 2009; File Size: 20.0KB.)

FIELD OF THE INVENTION

The present invention relates to methods for modulating IL-33 activity.

BACKGROUND OF THE INVENTION

The immune system protects individuals from infective agents, e.g.,bacteria, multi-cellular organisms, as well as cancers. This systemincludes several types of lymphoid and myeloid cells such as monocytes,macrophages, dendritic cells (DCs), eosinophils, T cells, B cells, andneutrophils. These lymphoid and myeloid cells often produce signalingproteins known as cytokines. Immune response includes inflammation,i.e., the accumulation of immune cells systemically or in a particularlocation of the body. In response to an infective agent or foreignsubstance, immune cells secrete cytokines which, in turn, modulateimmune cell proliferation, development, differentiation, or migration.Immune response sometimes results in pathological consequences, that is,inflammatory disorders. These inflammatory disorders, which involveimmune cells and cytokines, include, e.g., psoriasis, rheumatoidarthritis (RA), Crohn's disease (CD), multiple sclerosis (MS), andatherosclerosis.

The interleukin-1 (IL-1) family of cytokines contributes to thepathology of inflammatory disorders and proliferative conditions, e.g.,arthritis and cancer. There are 11 members of the IL-1 cytokine family.IL-1 cytokines bind to members of the IL-1 cytokine receptor family.There are ten members in the IL-1 receptor family, and IL-1 ligandstypically require participation of two different IL-1 receptors to makeup the cell surface complex. For example, IL-1α and IL-1β bind first tothe cell surface receptor IL-1R1, and this IL-1/IL-1R1 complexsubsequently binds to a second cell surface IL-1R family member, IL-1Receptor accessory chain protein (IL-1RAcP) Binding of IL-1α or IL-1β toboth receptor components is necessary to transduce the IL-1 signal.

IL-1 family members play a role in inflammatory conditions, e.g.,rheumatoid arthritis, psoriasis, asthma, chronic obstructive pulmonarydisorder (COPD), sepsis, and inflammatory bowel disorder (IBD).Rheumatoid arthritis (RA) is a common chronic inflammatory disordercharacterized by degradation of joints, e.g., the synovial membrane,cartilage, and bone. IL-1 stimulates a number of cells involved inarthritic inflammation, e.g., fibroblasts, osteoclasts, chondrocytes,and neutrophils, which may show abnormal proliferation and releaseenzymes causing joint destruction.

Proliferative disorders are the second most common cause of death in theUnited States. Cytokines of the IL-1 family have been implicated in thecontrol and pathology of proliferative disorders, i.e., cancer. IL-1modulates progression through the cell cycle, e.g., by changingexpression of cyclin-dependent kinases and cyclin-dependent kinaseinhibitors. High doses of IL-1β promote tumor invasiveness, while lowdoses can promote immune eradication of tumors.

IL-33 exerts its biological effects via the receptor protein ST2 andinduces T Helper Type 2-associated cytokines (Schmitz, J. et al.,Immunity 23: 479-490 (2005)). In cells stimulated with IL-33, thesignaling components MyD88, IRAK, IRAK4 and TRAF6 are recruited to ST2.In addition, IL-33 mediated signaling involves phosphorylation of IκBαand phosphorylation of the MAP kinases Erk1/2, p38 and JNK. T_(H)2cells, but not T_(H)1 cells, respond to IL-33 stimulation with increasedproduction of IL-5 and IL-13. IL-33 administration results insplenomegaly with significantly higher numbers of spleen eosinophils,mononuclear cells, and plasma cells, but not neutrophils. IL-33administration also results in increased levels of blood eosinophils,lymphocytes and neutrophils. IL-33 administration also leads toinduction of IL-4, IL-5 and IL-13 gene and protein expression in vivo.IL-33 administration also results in airway epithelial lininghypertrophy and mucous production, epithelial hyperplasia in theesophagus, and inflammatory infiltrates of eosinophils, neutrophils andmononuclear cells in the esophageal epithelium. In addition, IL-33administration results in increased levels of serum IgE and IgA.

There remains an unmet need to treat inflammatory and immune disorders.Specifically, the need exists for improved compositions and methods oftreatment for immune disorders related to IL-33 signaling in whichreduction of IL-33 activity would provide therapeutic benefit.

SUMMARY OF THE INVENTION

We have previously shown that the orphan receptor ST2 is one of thecomponents of the IL-33 receptor complex (Schmitz, J. et al., Immunity23: 479-490 (2005)). It was thought that the receptor complex that bindsIL-33 is a complex of ST2 and SIGIRR. See WO 2005/079844. Here, weexplain that the second component of the IL-33 receptor complex is notSIGIRR, but is instead IL-1RAcP. Although no receptor promiscuity hasbeen observed for the major IL-1 ligands (IL-1α and IL-1β exclusivelybind to IL-1R1 and IL-1RAcP, and IL-18 binds exclusively to IL-18Rα andIL-18Rβ), we focused on IL-1RAcP after we were unsuccessful in findingthe second IL-33 receptor among the orphan IL-1 receptors. Accordingly,the present invention provides that IL-33 signal transduction occursthrough ST2 and IL-1RAcP.

In one aspect, the present invention provides methods of modulating(e.g. preventing, reducing or ameliorating) an immune disorder orcondition, comprising inhibiting IL-33 signal transduction through ST2and IL-1RAcP by administering to a subject in need thereof an effectiveamount of: (a) an antagonist of IL-33 binding to a complex of ST2 andIL-1RAcP, and/or (b) an antagonist of IL-1RAcP binding to a complex ofIL-33 and ST2.

The present invention also provides methods of modulating blood cellcounts, comprising inhibiting IL-33 signal transduction through ST2 andIL-1RAcP by administering to a subject in need thereof an effectiveamount of: (a) an antagonist of IL-33 binding to a complex of ST2 andIL-1RAcP, and/or (b) an antagonist of IL-1RAcP binding to a complex ofIL-33 and ST2. Such modulation, e.g. may include increasing the count ofplatelets and/or decreasing the counts of one or more of total whiteblood cells, neutrophils, lymphocytes and eosinophils.

In various embodiments the antagonist comprises an antibody, orantigen-binding fragment thereof, that binds to IL-33, ST2, or a complexof IL-33 bound to ST2.

In other embodiments, the antagonist comprises an antibody, orantigen-binding fragment thereof, that binds to IL-1RAcP, a complex ofIL-1RAcP and ST2, or a complex of IL-1RAcP, ST2 and IL-33. In someembodiments the antibody or fragment does not bind to IL-33 alone and/ordoes not bind to ST2 alone.

In yet another embodiment, the antibody or fragment does not bind toIL-1RAcP alone.

In one embodiment, the antibody or fragment thereof is a monoclonal,humanized, or fully human antibody. In another embodiment, the antibodyor fragment is an Fab, an Fv fragment, or an F(ab′)₂ fragment.

In some embodiments, the immune disorder or condition is selected fromthe group consisting of an innate response, asthma, an allergy, multiplesclerosis, inflammatory bowel disorder, arthritis, an infection, cancer,and a tumor. Exemplary arthritic conditions may be selected from thegroup consisting of rheumatoid arthritis, osteoarthritis, and psoriaticarthritis. Exemplary infections may be selected from the groupconsisting of an intracellular pathogen, a bacterium, a parasite and avirus. Exemplary intracellular pathogens may be selected from the groupconsisting of Leishmania sp., Mycobacterium sp., Listeria sp.,Toxoplasma sp., Schistosoma sp.

In other embodiments, the immune disorder or condition is a T_(H)1-typeresponse or a T_(H)2-type response.

In another aspect, the present invention provides methods and kits forthe diagnosis of an immune condition or disorder. In one embodiment, thediagnostic method comprises determining the presence or level of one ormore of IL-33, ST2, IL-1RAcP, a complex of IL-33 and ST2, a complex ofST2 and IL-1RAcP and a complex of ST2, IL-1RAcP and IL-33 in a samplefrom a subject, and comparing that level to the levels in non-diseasedtissues, subjects that are known to have the immune condition ordisorder, and/or subjects that are known not to have the immunecondition or disorder. In one embodiment, the presence or level of acomplex of IL-33 and ST2 is detected. In one embodiment the detectioninvolves use of a binding compound comprising a detectable label, e.g.an antibody or antigen-binding fragment thereof. In another embodiment,the invention involves kits to perform the diagnostic methods of theinvention, comprising: a compartment; a detectably-labeled molecule,such as an antibody, that binds one or more of IL-33, ST2, IL-1RAcP, acomplex of IL-33 and ST2, a complex of ST2 and IL-1RAcP and a complex ofST2, IL-1RAcP and IL-33; and, optionally, instructions for use. In oneembodiment, the detectably-labeled molecule binds to a complex of IL-33and ST2.

In another aspect, the present invention provides an isolated andpurified complex of ST2 and IL-1RAcP, optionally further comprisingIL-33, and sequence variants thereof. In one embodiment the ST2,IL-1RAcP and IL-33 are human proteins. In other embodiments, theinvention provides such complexes comprising polypeptide having 70%,80%, 90%, 95%, 98% or 100% sequence identity with a naturally occurringhuman ST2, IL-1RAcP or IL-33 protein. In one aspect, the inventionprovides methods of making these isolated and purified complexes,comprising mixing two or more of the proteins and allowing said complexto form.

In yet another aspect, the present invention provides in vitro methodsof determining whether a test compound is an antagonist of IL-33 bindingto a complex of ST2 and IL-1RAcP, and/or IL-1RAcP binding to a complexof IL-33 and ST2, comprising an assay selected from the group consistingof a) an NF-κB-dependent reporter gene expression assay, b) an MyD88IRAK, IRAK4 or TRAF6 recruitment assay, c) an Erk1/2, p38, IκBα or JNKphosphorylation assay, d) an NF-κB, Erk1/2 or p38 phosphorylation assayin cells that naturally express ST2, e) an IL-13, IL-6 or IL-5expression assay, and f) an IL-6 production assay in mouse mast cellline WTMC. In some embodiments, the test compound is determined to be anantagonist if it reduces the activity of IL-33 in the assay whencompared to an assay run without the test compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that IL-33 administration increases the percentage ofeosinophils found in the blood of wild-type (WT) mice, but not IL-1RAcPdeficient (KO) mice. Individual data bars represent separateexperimental animals, with one experiment involving five animals (FIG.1A) and another involving three animals (FIG. 1B).

FIG. 2 shows that IL-33 administration increases IL-5 production inserum (FIG. 2A) and bronchoalveolar lavage (BAL) (FIG. 2B) in wild-type(WT) mice, but not in IL-1RAcP deficient (KO) mice.

FIG. 3 shows that IL-33 administration increases serum IgE in wild-type(WT) mice, but not in IL-1RAcP deficient (KO) mice.

FIGS. 4A-4F show that, unlike wild-type (WT) mice, IL-1RAcP deficient(KO) mice do not upregulate T_(H)2 cytokine or cytokine receptor genesin response to IL-33 administration

FIG. 5 shows that IL-33 induces IL-6 production from WTMC cells in adose dependent manner.

FIG. 6 shows that blocking ST2 prevents IL-33-induced IL-6 production inWTMC cells.

FIG. 7 shows that IL-1RAcP is necessary for IL-33-enhanced IL-13 (FIG.7A), IL-6 (FIG. 7B), and IL-5 (FIG. 7C) production from T_(H)2 cells

FIG. 8 shows the detection of the ST2/IL-33/IL-1RAcP complex, in whichST2 is immobilized on the plate in FIG. 8A and IL-1R1 is immobilized onthe plate in FIG. 8B.

FIG. 9 shows that dominant negative IL-1RAcP inhibits IL-33 signaling.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Administering” refers to the delivery to an animal, human, subject,cell, tissue, organ, or biological fluid of an exogenous composition,pharmaceutical, therapeutic, or diagnostic agent, e.g. an agentcontaining an antagonist of IL-33 signal transduction through ST2 andIL-1RAcP. In one embodiment, “administering” refers to delivery to asubject. In another embodiment, “administering” refers to delivery to ahuman subject.

An “antagonist of IL-33 binding to a complex of ST2 and IL-1RAcP” is amolecule that inhibits IL-33 binding to a complex of ST2 and IL-1RAcP.An “antagonist of IL-1RAcP binding to a complex of IL-33 and ST2” is amolecule that inhibits binding of IL-1RAcP to a complex of IL-33 andST2. In one embodiment, the antagonist is an antibody, such as apolyclonal antibody, a monoclonal antibody, a humanized antibody, or ahuman antibody. In another embodiment, the antagonist is an antibodyfragment, such as a Fab, an Fv fragment, or a F(ab′)₂ fragment.

In another embodiment, the antibody or fragment thereof specificallybinds to (a) IL-33, (b) IL-1RAcP, (c) ST2, (d) a complex of ST2 bound toIL-1RAcP, (e) a complex of IL-33 bound to ST2, or (f) a complex ofIL-33, ST2 and IL-1RAcP.

In one embodiment, an “effective amount” of the antagonist of IL-33signal transduction through ST2 and IL-1RAcP means an amount sufficientto ameliorate a symptom or sign of a disorder or physiologicalcondition, or an amount sufficient to permit or facilitate the diagnosisof a disorder or physiological condition. An effective amount for ahuman or veterinary subject may vary depending on factors such as thecondition being treated, the overall health of the subject, the methodroute and dose of administration and the severity of side affects. Inone embodiment, an effective amount is the maximal dose or dosingprotocol that avoids significant side effects or toxic effects. Theeffect will result in an improvement of a diagnostic measure, parameter,or detectable signal by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or 90%, where 100% is defined as the diagnostic parameter shown bya normal subject (see, e.g., Maynard, et al., A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, Fla. (1996); Dent,Good Laboratory and Good Clinical Practice, Urch Publ., London, UK(2001)).

In another embodiment, an “effective amount” of the antagonist of IL-33signal transduction through ST2 and IL-1RAcP means an amount sufficientto inhibit IL-33 signal transduction through ST2 and IL-1RAcP.

“Inhibiting IL-33 signal transduction through ST2 and IL-1RAcP” meansthat the degree to which IL-33 stimulates signal transduction throughST2 and IL-1RAcP is diminished in the presence of an antagonist,relative to the degree to which IL-33 stimulates signal transductionthrough ST2 and IL-1RAcP in the absence of the antagonist.

To examine the extent of inhibition, a sample is treated with apotential inhibitor and is compared to a control sample without theinhibitor. Control samples, i.e., not treated with antagonist, areassigned a relative activity value of 100%. Inhibition is achieved whenthe activity value relative to the control is about 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% or less.

An endpoint in inhibition may comprise a predetermined quantity orpercentage of, e.g., an indicia of inflammation, oncogenicity, or celldegranulation or secretion, such as the release of a cytokine. Anendpoint of inhibition is generally 75%, 50%, 25%, or 10% of the controlor less.

Inhibition of IL-33 signal transduction through ST2 and IL-1RAcP can bedetermined by assaying for IL-33 signal transduction in an in vitroassay. One in vitro assay that can be used is an NF-κB-dependentreporter assay (Schmitz, J. et al., Immunity 23: 479-490 (2005)). Inthis assay, cells, e.g., HEK293FT cells, are transfected with either amock expression vector or with an expression vector encoding ST2 inconjunction with an NF-κB-driven reporter gene expression product, e.g.,green fluorescent protein (GFP). Cells are incubated with IL-33 alone,or with IL-33 and a test molecule that is being tested for antagonisticactivity. Cells are then analyzed for reporter gene expression product,e.g., by fluorescence activated cell sorting (FACS®). If IL-33 treatedcells exhibit reduced reporter gene expression after incubation with thetest molecule, the test molecule is an antagonist of IL-33 binding tothe IL-1RAcP receptor complex.

Another reporter gene expression product that can be used is luciferase.In this assay, cellular extracts are assayed for NF-κB-driven luciferaseexpression in response to incubation with IL-33, and with or without atest molecule, i.e. a putative inhibitor of IL-33 signal transduction.

Another in vitro assay that can be performed is to assay for MyD88 IRAK,IRAK4, or TRAF6 recruitment (Schmitz, J. et al., Immunity 23: 479-490(2005)). In this assay, cells, e.g., HEK293FT cells, are transfectedwith either a mock expression vector or with an expression vectorencoding ST2, and incubated with IL-33. In the control experiment, notest molecule is added to the incubation. In the test experiment, themolecule to be tested for its ability to inhibit IL-33 signaltransduction is added. Cellular extracts are immunoprecipitated with ananti-ST2 antibody, followed by Western analysis with anti-MyD88, IRAK,IRAK4, or TRAF6 antibodies. A decrease in expression of MyD88, IRAK,IRAK4, or TRAF6 in extracts from IL-33 treated cells when incubated withthe test molecule indicates that the test molecule is an antagonist ofIL-33 binding to the IL-1RAcP receptor complex.

Another in vitro assay that can be performed is to assay forphosphorylation of Erk1/2, p38, IκBα, or JNK (Schmitz, J. et al.,Immunity 23: 479-490 (2005)). In this assay, cells, e.g., HEK293FTcells, are transfected with either a mock expression vector or with anexpression vector encoding ST2, and incubated with IL-33. In the controlexperiment, no test molecule is added to the incubation. In the testexperiment, the molecule to be tested for its ability to inhibit IL-33signal transduction is added. Cellular extracts are prepared and blottedwith anti-phospho-Erk1/2, anti-phospho-p38, anti-phospho-IκBα, oranti-phospho-JNK antibodies. A decrease in the level of phosphorylatedErk1/2, p38, IκBα, or JNK in extracts from IL-33 treated cells whenincubated with the test molecule indicates that the test molecule is anantagonist of IL-33 binding to the IL-1RAcP receptor complex.

Another in vitro assay that can be performed is to assay forphosphorylation of NF-κB, Erk1/2 or p38 in cells that naturally expressST2, e.g., mouse mast cells (WTMC) (Schmitz, J. et al., Immunity 23:479-490 (2005)). In the control experiment, WTMC cells are incubatedwith IL-33 but no test molecule is added to the incubation. In the testexperiment, cells are incubated with IL-33 and the molecule to be testedfor its ability to inhibit IL-33 signal transduction. Cell lysates areseparated by SDS-PAGE and electroblotted. Cellular extracts are blottedwith anti-NF-κB, anti-phospho-Erk1/2, or anti-phospho-p38 antibodies. Adecrease in the level of phosphorylated NF-κB, Erk1/2, or p38 inextracts from IL-33 treated cells when incubated with the test moleculeindicates that the test molecule is an antagonist of IL-33 binding tothe IL-1RAcP receptor complex.

Another in vitro assay that can be performed is to assay expression ofIL-5 or IL-13 in T_(H)2 cells (Schmitz, J. et al., Immunity 23: 479-490(2005)). In the control experiment, T_(H)2 cells are incubated withIL-33 but no test molecule is added to the incubation. In the testexperiment, T_(H)2 cells are incubated with IL-33 and the molecule to betested for its ability to inhibit IL-33 signal transduction.Supernatants are analyzed for IL-5 and IL-13 production. A decrease inthe level of IL-5 or IL-13 in supernatant from IL-33 treated cells whenincubated with the test molecule indicates that the test molecule is anantagonist of IL-33 binding to the IL-1RAcP receptor complex.

Another in vitro assay that can be used to determine whether a moleculeis an antagonist of IL-33 binding is to determine the effect of a testmolecule on IL-33-induced production of IL-6 in the mouse mast cell lineWTMC. A decrease in the level of IL-6 production by IL-33 treated cellswhen incubated with the test molecule indicates that the test moleculeis an antagonist of IL-33 binding to the IL-1RAcP receptor complex.

In vivo assays that can be used to determine whether a molecule is anantagonist of IL-33 binding to the IL-1RAcP receptor complex is todetermine the effect of a test molecule on the influence of IL-33administration on spleen size, spleen cells (e.g., mononuclear cells,eosinophils, and plasma cells), blood cell count (e.g., eosinophils,lymphocytes or neutrophils) (Schmitz, J. et al., Immunity 23: 479-490(2005)). A decrease in the effect of IL-33 after administration of IL-33and the test molecule on an in vivo endpoint, relative to the effect ofIL-33 alone, indicates that the test molecule is an antagonist of IL-33binding to the IL-1RAcP receptor complex.

In a one embodiment, the antagonist that is assayed for in theabove-described assays is an antibody.

“Expression” refers to a measure of mRNA or polypeptide encoded by aspecific gene. Units of expression may be a measure of, e.g., the numberof molecules of mRNA or polypeptide/mg protein, the number of moleculesof mRNA or polypeptide/cell, in measurements of expression by cell,tissue, cell extract, or tissue extract. The units of expression may berelative, e.g., a comparison of signal from control and experimentalmammals or a comparison of signals with a reagent that is specific forthe mRNA or polypeptide versus with a reagent that is non-specific.

“Immune condition” or “immune disorder” encompasses, e.g., pathologicalinflammation, an inflammatory disorder, and an autoimmune disorder ordisease. “Immune condition” also refers to infections, persistentinfections, and proliferative conditions, such as cancer, tumors, andangiogenesis.

In one embodiment, the immune disorder or immune condition is selectedfrom the group consisting of an innate response, asthma, allergy,multiple sclerosis, inflammatory bowel disorder, arthritis, aninfection, cancer, and a tumor. In another embodiment, the arthritis isselected from the group consisting of rheumatoid arthritis,osteoarthritis, and psoriatic arthritis. In another embodiment, theimmune disorder or condition comprises a T_(H)1-type response or aT_(H)2-type response.

“Infection” refers to a disorder resulting from a pathogen, microbe,bacterium, parasite, virus, and the like. In one embodiment, theinfection is caused by an organism selected from the group consisting ofa bacterium, a parasite, and a virus. In another embodiment, theinfection is caused by an organism selected from the group consisting ofLeishmania sp., Mycobacterium sp., Listeria sp., Toxoplasma sp.,Schistosoma sp., and a respiratory virus.

“Inflammatory disorder” means a disorder or pathological condition wherethe pathology results, in whole or in part, from, e.g., a change innumber, change in rate of migration, or change in activation, of cellsof the immune system. Cells of the immune system include, e.g., T cells,B cells, monocytes or macrophages, antigen presenting cells (APCs),dendritic cells, microglia, NK cells, NKT cells, neutrophils,eosinophils, mast cells, or any other cell specifically associated withthe immunology, for example, cytokine-producing endothelial orepithelial cells.

“IL-1 RAcP” means the Interleukin-1 Receptor Accessory Protein. IL-1RAcP is further described at online Mendelian Inheritance in Man (OMIM)entry 602626 and Gene ID No. 3556, and exemplary naturally occurringsequences for human IL-1 RAcP are found at GenBank Accession Nos.NP_(—)608273 (SEQ ID NO: 1) and NP_(—)002173 (SEQ ID NO: 2), all ofwhich are available through the National Center for BiotechnologyInformation (NCBI) website.

“IL-33 receptor complex” refers to the specific binding of ST2 toIL-1RAcP to form a protein complex that itself specifically binds toIL-33 and transduces the IL-33 signal from the cell surface to the cellinterior.

“IL-33/ST2 complex” refers to the specific binding of IL-33 and ST2 toform a protein complex that itself specifically binds to IL-1 RAcP.IL-33 is further described at OMIM entry 608678 and Gene ID No. 90865,and an exemplary naturally occurring sequence for human IL-33 (SEQ IDNO: 3) is found at GenBank Accession No. AY905581, all of which areavailable through the NCBI website. See also Schmitz et al. (2005)Immunity 23: 479-490.

An “isolated and purified” complex, such as an isolated and purifiedcomplex of ST2 and IL-1RAcP, optionally further comprising IL-33, is anin vitro complex that is isolated and purified from its natural source.Specifically, an isolated and purified complex does not encompasscomplexes as they form in nature. Such complexes may be formed, e.g., bymixture of two or more purified proteins, or by mixture of one purifiedprotein with a cell extract or biological sample, or by the purificationof a complex from a natural source.

“IL-33 signal transduction through ST2 and IL-1RAcP” refers to themolecular events that occur when IL-33, ST2 and IL-1RAcP form a proteincomplex. In one embodiment, the IL-33 signal transduction refers torecruitment of one or more of MyD88, IRAK, IRAK4, and TRAF6 to ST2 inresponse to the formation of a complex of IL-33, ST2, and IL-1RAcP. Inanother embodiment, IL-33 signal transduction refers to phosphorylationof one or more of IκBκ, Erk1/2, p38 and JNK in response to the formationof a complex of IL-33, ST2, and IL-1RAcP. In another embodiment, IL-33signal transduction refers to IL-5 or IL-13 secretion by T_(H)2 cells.

Specificity of binding refers to a binding interaction between apredetermined ligand and a predetermined receptor that enables one todistinguish between the predetermined ligand and other ligands, orbetween the predetermined receptor and other receptors. In oneembodiment, the ligand is IL-33, and the receptor is ST2. In anotherembodiment, the ligand is IL-33, and the receptor is a protein complexof ST2 and IL-1RAcP. In another embodiment, the ligand is a proteincomplex of IL-33 and ST2, and the receptor is IL-1RAcP. In anotherembodiment, the ligand is an antibody or a fragment thereof thatspecifically binds to IL-33, IL-1RAcP, ST2, a complex of ST2 bound toIL-1RAcP, a complex of IL-33 bound to ST2, or a complex of IL-33, ST2and IL-1RAcP.

A “pharmaceutical composition” comprises the active drug ingredient andone or more pharmaceutically acceptable excipients, carriers and/ordiluents.

“Specifically binds” means a binding reaction that is determinative ofthe presence of a protein in a heterogeneous population of proteins andother biologics. For example, under designated conditions, one proteinbinds to another protein and does not bind in a significant amount toother proteins present in the sample. In various embodiments, anantibody or fragment thereof binds to its antigen with an affinity thatis at least two fold greater, at least ten times greater, at least20-times greater, or at least 100-times greater than the affinity to anyother protein.

In various embodiments, an antibody, or fragment thereof, thatspecifically binds to IL-33, IL-1RAcP, ST2, a complex of ST2 bound toIL-1RAcP, a complex of IL-33 bound to ST2, or a complex of IL-33, ST2and IL-1RAcP will bind with a K_(D) of at least about 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, or 10⁻¹¹ M or better (lower K_(D)).

“ST2” as used herein is synonymous with the T1/ST2 protein (also knownas IL-1RL1). ST2 is further described at OMIM entry 601203 and Gene IDNo. 9173, and exemplary naturally occurring sequences for human ST2 arefound at GenBank Accession Nos. NP_(—)003847 (SEQ ID NO: 4) andNP_(—)057316 (SEQ ID NO: 5), all of which are available through the NCBIwebsite.

“ST2/IL-1RAcP complex” refers to the specific binding of ST2 andIL-1RAcP to form a protein complex that itself specifically binds IL-33.

Purification of antigen is not necessary for the generation ofantibodies. Immunization can be performed by DNA vector immunization,see, e.g., Wang, et al. Virology 228:278-284 (1997). Alternatively,animals can be immunized with cells bearing the antigen of interest.Splenocytes can then be isolated from the immunized animals, and thesplenocytes can fused with a myeloma cell line to produce a hybridoma(Meyaard et al., Immunity 7:283-290 (1997); Wright et al., Immunity13:233-242 (2000); Preston et al., Eur. J. Immunol. 27:1911-1918(1997)). Resultant hybridomas can be screened for production of thedesired antibody by functional assays or biological assays, that is,assays not dependent on possession of the purified antigen. Immunizationwith cells may prove superior for antibody generation than immunizationwith purified antigen (Kaithamana et al., J. Immunol. 163:5157-5164(1999)).

Any suitable method can be used to elicit an antagonist antibody withthe desired biologic properties. It may be desirable to preparemonoclonal antibodies (mAbs) from mammalian hosts such as mice, rodents,primates, humans, etc. Techniques for preparing such monoclonalantibodies may be found in, e.g., Stites et al. (eds.) BASIC ANDCLINICAL IMMUNOLOGY (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane (1988) ANTIBODIES:A LABORATORY MANUAL CSH Press; Goding (1986) MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. Thus,monoclonal antibodies may be obtained by a variety of techniquesfamiliar to researchers skilled in the art. Typically, spleen cells froman animal immunized with a desired antigen are immortalized, commonly byfusion with a myeloma cell. See Kohler and Milstein (1976) Eur. J.Immunol. 6:511-519. Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods known in the art. See, e.g., Doyle et al. (eds. 1994 andperiodic supplements) CELL AND TISSUE CULTURE: LABORATORY PROCEDURES,John Wiley and Sons, New York, N.Y. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a antigen binding fragment thereof byscreening a DNA library from human B cells according, e.g., to thegeneral protocol outlined by Huse et al. (1989) Science 246:1275-1281.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse et al. supra; and Ward etal. (1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced, see Cabilly U.S. Pat. No. 4,816,567; and Queen et al. (1989)Proc. Nat'l Acad. Sci. USA 86:10029-10033; or made in transgenic mice,see Mendez et al. (1997) Nature Genetics 15:146-156. See also Abgenixand Medarex technologies.

Any suitable non-human antibody can be used as a source for thehypervariable region of a humanized antibody. Sources for non-humanantibodies include, but are not limited to, murine, Lagomorphs(including rabbits), bovine, and primates. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichhypervariable region residues of the recipient are replaced byhypervariable region residues from a non-human species (donor antibody)such as mouse, rat, rabbit or nonhuman primate having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance of the desired biological activity. For further details, seeJones et al. (1986) Nature 321:522-525; Reichmann et al. (1988) Nature332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

Methods for recombinantly engineering antibodies have been described,e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly et al. (U.S.Pat. No. 4,816,567), Law et al. (European Patent Application PublicationNo. 438310) and Winter (European Patent Application Publication No.239400).

In one embodiment, the antagonist of the present invention isadministered in a pharmaceutical composition. In another embodiment, theantagonist of the present invention is administered in a sterilecomposition.

To prepare pharmaceutical or sterile compositions, an active drugingredient is mixed with one or more pharmaceutically acceptablecarriers, excipients and/or diluents. A Pharmaceutical formulation canbe prepared in the form of, e.g., a lyophilized powder, an aqueoussolution, or a suspension. See, e.g., Hardman, et al., Goodman andGilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y. (2001); Gennaro, Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y. (2000);Avis, et al. (eds.), Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY (1993); Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y. (2000).

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. Preferably,an administration regimen maximizes the amount of therapeutic deliveredto the patient consistent with an acceptable level of side effects.Accordingly, the amount of biologic delivered depends in part on theparticular entity and the severity of the condition being treated.Guidance in selecting appropriate doses of antibodies is available. See,e.g., Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd,Oxfordshire, UK (1996); Kresina (ed.), Monoclonal Antibodies, Cytokinesand Arthritis, Marcel Dekker, New York, N.Y. (1991); Bach (ed.),Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, MarcelDekker, New York, N.Y. (1993).

An antibody or fragment thereof can be provided by continuous infusion,or by doses at intervals. An antibody or fragment thereof may beadministered intravenously, intramuscularly, or subcutaneously.

A preferred dose protocol is one involving the maximal dose or dosefrequency that avoids significant undesirable side effects. In variousembodiments, the total weekly dose is 0.05 μg/kg, 0.2 μg/kg, 0.5 μg/kg,1 μg/kg, 10 μg/kg, 100 μg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg,25 mg/kg, 50 mg/kg or more.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside affects (see, e.g., Maynard, et al., A Handbook of SOPs for GoodClinical Practice, Interpharm Press, Boca Raton, Fla. (1996); Dent, GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK (2001)).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is derived from the same species as the animal targeted fortreatment, thereby minimizing a humoral response to the reagent.

The present invention also provides a method of modulating blood cellcounts, the method comprising administering an effective amount of anantagonist of IL-33 signal transduction through ST2 and IL-1RAcP. In oneembodiment, the antagonist increases the count of platelets. In anotherembodiment, the antagonist decreases the counts of total white bloodcells, neutrophils, lymphocytes, and/or eosinophils.

The present invention also provides methods and kits for the diagnosisof an immune condition or disorder. A method of diagnosis can comprisecontacting a sample from a subject, e.g., a test subject, with anantibody or fragment thereof that specifically binds to (a) IL-33, (b)IL-1RAcP, (c) ST2, (d) a complex of ST2 and IL-1RAcP, (e) a complex ofIL-33 and ST2, or (f) a complex of IL-33, ST2 and IL-1RAcP. The methodcan further comprise contacting a sample from a control subject, normalsubject, or normal tissue or fluid from the test subject, with anantibody or fragment thereof. Moreover, the method can additionallycomprise comparing the specific binding of the antibody or fragmentthereof to the test subject with the specific binding of the compositionto the normal subject, control subject, or normal tissue or fluid fromthe test subject. Expression or activity of a test sample or testsubject can be compared with that from a control sample or controlsubject. A control sample can comprise, e.g., a sample of non-affectedor non-inflamed tissue in a patient suffering from an immune disorder.Expression or activity from a control subject or control sample can beprovided as a predetermined value, e.g., acquired from a statisticallyappropriate group of control subjects.

Kits for the diagnosis of an immune condition or disorder may comprise acompartment and a binding compound, e.g. an antibody or antigen-bindingfragment thereof, that specifically binds to one or more components ofIL-33 signal transduction through ST2 and IL-1RAcP. Such kits mayoptionally include instructions for use. In some embodiments the bindingcompound is detectably labeled.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto specific embodiments.

Many modifications and variations of this invention, as will be apparentto one of ordinary skill in the art can be made to adapt to a particularsituation, material, composition of matter, process, process step orsteps, to preserve the objective, spirit and scope of the invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto without departing from the spirit and scope of theinvention. The specific embodiments described herein are offered by wayof example only, and the invention includes the full scope ofequivalents to which such claims are entitled; and the invention is notto be limited by the specific embodiments that have been presentedherein.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication, patent application, patent,database entry or other document was specifically and individuallyindicated to be incorporated by reference including all figures anddrawings.

EXAMPLE 1 Animal Experiments

In order to determine whether IL-1RAcP is the second member of thereceptor complex, wild-type (WT) and IL-1RAcP deficient (KO) mice wereused. The wild-type (WT) mice used were strain B6; 129SF2/J (JacksonLaboratories). The IL-1RAcP deficient (KO) mice used were strain B6;129S1-Il1rap^(tm1/Rom1)/J (Jackson Laboratories). The IL-1RAcP deficientmice have been genetically altered and do not have a functional IL-1RAcPreceptor protein.

Wild-type and IL-1RAcP deficient mice were given daily intraperitonealinjections of saline or 2 micrograms of human IL-33 for six days. On dayseven, the mice were sacrificed and analyzed for IL-33 responsiveness byseveral endpoints, as discussed in the following Examples. Bloodeosinophilia was measured in blood smears. Serum and bronchoalveolarlavage (BAL) fluid were collected to quantitate cytokine levels andserum immunoglobulin (Ig) E levels. Lung and spleen tissue was collectedto quantitate messenger RNA of key cytokines Lung and small intestinetissue was analyzed for histological changes. Two replicate experimentswere performed with either three or five mice per treatment group.

The following data show that IL-1RAcP deficient mice do not respond toIL-33 administration, indicating that IL-1RAcP is necessary to mediatethe in vivo effects of IL-33. These results implicate IL-1RAcP as thesecond component of the IL-33 receptor.

EXAMPLE 2 Lack of Blood Eosinophilia in IL-1RAcP Deficient Mice

Tail blood was smeared onto glass slides, fixed in 95% ethanol, andstained by the Wright-Giemsa method. Cells were enumerated bymicroscope, and at least 300 cells were counted per mouse. The graphs inFIGS. 1A and 1B represent the two replicate experiments performed,involving five and three animals, respectively. IL-33 administration towild-type mice dramatically increased the percentage of eosinophilsfound in the blood. However the percent of eosinophils in the blood ofIL-33-treated IL-1RAcP deficient mice was not increased, and was similarto saline treated mice.

EXAMPLE 3 Lack of Goblet Cell Hyperplasia and Mucous Production inIL-1RAcP Deficient Mice

Formalin-fixed, paraffin-embedded small intestines were stained byPeriodic acid-Schiff. Wile-type mice, but not IL-1RAcP deficient mice,responded to IL-33 with increased goblet cell mucous production in thesmall intestine. A total of eight mice were examined per treatmentcondition. A similar finding was noted in the upper airways of the lung.

EXAMPLE 4 IL-1RAcP Deficient Mice Fail to Respond to IL-33 withIncreased Production of IL-5

Serum and BAL fluid were analyzed for cytokine content by multipleximmunoassay. In both the serum (FIG. 2A) and BAL fluid (FIG. 2B), IL-33treatment dramatically increased the production of IL-5 in wild-typemice, but not in IL-1RAcP deficient mice. The data are representative oftwo experiments.

EXAMPLE 5 Serum Levels of IgE are Increased in Wild-Type but notIL-1RAcP Deficient Mice

Serum IgE levels were measured by ELISA. IL-33 increased serum IgE inwild-type, but not in IL-1RAcP deficient mice (FIG. 3). The data arerepresentative of two experiments.

EXAMPLE 6 IL-1RAcP Deficient Mice do not Upregulate T_(H)2Cytokine Genesin Response to IL-33 Administration

RT-PCR was performed on RNA obtained from snap-frozen spleen and lungtissue, and gene expression is shown in FIG. 4, relative to the levelsof ubiquitin (ub) in the sample. IL-33 increased mRNA expression of theT helper 2 (T_(H)2) cytokines and cytokine receptors IL-4 (FIG. 4A),IL-5 (FIG. 4C), IL-6 (FIG. 4B), IL-13 (FIG. 4D), IL-17RB (FIG. 4E), andST2 (FIG. 4F), in the lung and spleen of wild-type mice, but not inIL-1RAcP deficient (“KO”) mice.

EXAMPLE 7 IL-33 Induces IL-6 Production in WTMC Cells

The mouse mast cell line WTMC (Wright, G. J., et al., J. Immunol. 171:3034-3046 (2003)), expresses ST2 and responds to both human and mouseIL-33 in a dose dependent manner by producing IL-6. IL-33 activity canbe blocked by preincubating the cells with a blocking rat anti-mouse ST2antibody (MD Biosciences).

1×10⁵ WTMCs were plated per well (96-well plate format) in RPMI 1640w/L-glutamine containing 10% fetal calf serum, 1 mM Na-Pyruvate, 0.05 mM2-mercapto-ethanol, 0.2 mM L-glutamine, 1×MEM non-essential amino acids,1× PenStrep, 2.5 ng/ml mouse IL-3, and 2.5 ng/ml mouse IL-4. Cells wereincubated for 24 hours with varying concentrations of mouse or humanIL-33. Supernatants were assessed for IL-6 production by ELISA. As shownin FIG. 5, IL-33 induced IL-6 production in a dose dependent manner.

As shown in FIG. 6, a blocking rat anti-mouse ST2 antibody (MDBiosciences) prevented IL-33 induced production in WTMCs. Mast cellswere seeded with into 24 well plates at 1×10⁶ cells/well. Some cellswere preincubated with anti-ST2 antibody (10 μg/ml) prior to theaddition of IL-33 (50 ng/ml). Supernatants were collected after 6 and 24hours and analyzed for cytokine production by multiplex immunoassay.

EXAMPLE 8 IL-1RAcP is Necessary for IL-33-Enhanced IL-5, IL-6, and IL-13Production from T_(H)2Cells

CD4+ T cells were isolated from wildtype (B6; 129SF2/J) and IL-1RAcPknock-out (B6; 129S1-Il1rap^(tm1/Rom1)/J) mouse spleen and lymph nodesusing the CD4+ T cell isolation kit (Miltenyi Biotec). To polarizetowards a T_(H)2 phenotype, cells were cultured on anti-CD3 coatedplates in the presence of 5 ng/ml IL-2, 10 ng/ml IL-4, 10 μg/mlanti-IFNγ, and 1 μg/ml anti-CD28 in complete RPMI for 4 days. Cells werethen washed, and rested for 3 days in IL-2 (5 ng/ml) alone. Cells werethen washed and stimulated with media alone, human IL-33 (50 ng/ml), ormouse IL-1β (10 ng/ml). Some cells were preincubated with anti-ST2blocking antibody prior to cytokine addition. Supernatants wereharvested after 24 hours and cytokine levels were measured using amultiplexed immunoassay (22-plex mouse cytokine premixed beads, Linco),and analyzed by Luminex® immunoassay.

As shown in FIG. 7, both IL-1β and IL-33 enhanced the production ofIL-13, IL-6, and IL-5 in wildtype, but not IL-1RAcP−/− T_(H)2 cells. Theincreased production of these cytokines in wildtype mice by IL-33 wasblocked in the presence of anti-ST2 antibodies. Anti-ST2 did not blockIL-1β-enhanced cytokine production.

EXAMPLE 9 Detection of the ST2/IL-33/IL-1RAcP Complex

Murine IL-1β or IL-33 was titrated into plates containing solublehis6-tagged murine IL-1RAcP and plate-bound ST2-Fc (FIG. 8A) orIL-1R1-Fc (FIG. 8B). Peroxidase-conjugated anti-his6 was used as adetection antibody. Data is representative of 2 experiments. Datarepresent the mean+/−SEM of duplicate wells.

Receptor-ligand complex ELISA: 3 μg/mlmurine ST2-Fc or IL-1R1-Fc fusionproteins (R&D Systems) in PBS were coated overnight onto MaxiSorp plates(Nunc). After washing, plates were incubated with 3 μg/ml recombinanthis6-tagged murine IL-1RAcP and increasing concentrations of murineIL-33 or IL-1β in PBS/1% bovine serum albumin/0.05% Tween-20®polyoxyethylene sorbitan monolaurate for 1.5 hours. Plates were washedand incubated with anti-his6-peroxidase (Roche Diagnostics) for 1 hour,washed again, developed with TMB Peroxidase substrate (KPL), stoppedwith H₂PO₄, and read on a plate reader at 450-570 nm.

Although IL-1RAcP is required to mediate the effects of IL-33 in vivoand on Th2 cells, the possibility exists that this is due to an indirectrather than a direct interaction of IL-33 and IL-1RAcP. Therefore, wewished to detect formation of the ligand-receptor complex in vitro. Inthe IL-1 receptor complex, IL-1RAcP cannot bind IL-1 directly, rather itis hypothesized to interact with the complex of IL-1 and its primaryreceptor, IL-1R1. We are also unable to see a direct interaction ofIL-33 with IL-1RAcP (not shown). However, we can detect a specificST2/IL-33/IL-1RAcP complex by ELISA. We titrated IL-33 or IL-1β intowells containing plate-bound ST2 and soluble his6-tagged IL-1RAcP (FIG.8A). We detect IL-33 receptor complex formation with an anti-his6antibody as the concentration of IL-33, but not IL-1β, increases.Conversely, if the plates are coated with IL-1R1 instead of ST2, wedetect IL-1 receptor complex formation with increasing amounts of IL-1βbut not IL-33 (FIG. 8B). This result demonstrates that ST2, IL-33, andIL-1RAcP can form a ligand/receptor complex.

EXAMPLE 10 Dominant Negative IL-1RAcP Inhibits IL-33 Signaling

Signal transduction by IL-1RAcP in the IL-1 signaling cascade ismediated by its cytoplasmic Toll/IL-1 receptor (TIR) domain and involvesrecruitment of the adaptor MyD88 and activation of NF-κB. IL-33 uses thesame signaling components as IL-1, including IRAK, IRAK4, MyD88, andTRAF6, leading to the activation of NF-κB and MAP kinases. MyD88 isrequired for IL-33 signaling, as MyD88−/− mice do not respond to IL-33administration (data not shown). In order to investigate thecontribution of IL-1RAcP to IL-33 signal transduction, we used adominant negative form of IL-1RAcP that contains a stop codon before theTIR domain.

As a positive control, HEK293FT cells, which endogenously express humanIL-1R1 and IL-1RAcP, were transfected with 3 μg of a NF-κB-drivenreporter gene construct (pNF-κB-hrGFP, Stratagene), and with increasingamounts of cMyc-tagged murine dominant negative (dn) IL-1RAcP construct.Cells were stimulated at 24 hours post-transfection with 20 ng/ml murineIL-1α (R&D Systems) for 24 hours, and analyzed for GFP expression byFACS® cell sorting. This control tests the ability of the dn IL-1RAcP tointeract with the endogenous IL-1R1 and block IL-1α mediated downstreamsignaling. This interaction is reflected by the diminution of GFPexpression when increasing amounts of the dn IL1-RAcP are transfectedinto control cells (FIG. 9, bottom row).

For experimental samples, HEK293FT cells were transfected with 1 μg of aplasmid encoding murine ST2, 3 μg of the NF-KB-driven reporter geneconstruct, and with increasing amounts of cMyc-tagged murine dominantnegative (dn) IL-1RAcP construct. Cells were stimulated at 24 hourspost-transfection with 20 ng/ml murine IL-33 for 24 hours, and analyzedfor GFP expression by FACS® cell sorting. Results of a typicalexperiment are provided at FIG. 9 (top row). This experimental systemtests the ability of the dn IL-1RAcP to interact with murine ST2 andblock IL-33 signaling. The addition of the dn IL-1RAcP caused a roughlydose-responsive decrease in IL-33-induced reporter gene expression,suggesting that ST2 uses IL-1RAcP as a second receptor to mediate IL-33signal transduction.

1. A method of treating asthma or allergy, comprising inhibiting IL-33signal transduction by administering to a subject in need thereof aneffective amount of an antibody, or antigen binding fragment thereof,that specifically binds to a complex of ST2 (residues 19-556 of SEQ IDNO: 5) and IL-1 RAcP (residues 21-570 of SEQ ID NO: 2).
 2. The method ofclaim 1, wherein the antibody or fragment thereof does not bind to ST2alone.
 3. The method of claim 1, wherein the antibody or fragmentthereof is a humanized antibody or human antibody.
 4. The method ofclaim 1, wherein the antibody or fragment thereof is a fragment selectedfrom the group consisting of a Fab, an Fv fragment, and an F(ab′)2fragment.
 5. A method of decreasing the count of eosinophils, comprisinginhibiting IL-33 signal transduction by administering to a subject inneed thereof an effective amount of an antibody, or antigen bindingfragment thereof, that specifically binds to a complex of ST2 (residues19-556 of SEQ ID NO: 5) and IL-1 RAcP (residues 21-570 of SEQ ID NO: 2).6. The method of claim 5, wherein the antibody or fragment thereof is ahumanized antibody or human antibody.
 7. The method of claim 5, whereinthe antibody or fragment thereof is a fragment selected from the groupconsisting of a Fab, an Fv fragment, and an F(ab′)2 fragment.